Previously, we selected mutants of human rotavirus (HRV) serotypes of epidemiologic importance during successive serial passages in primary AGMK cells at progressively lower suboptimal temperatures (30 degrees Celsius, 28 degrees Celsius, and 26 degrees Celsius). These cold-adapted mutants include strain D (VP4:1A; VP7:1), DS-1 (VP4:1B; VP7:2), Wa x DS-1 (VP4:1A; VP7:2), Wa x P (VP4:1A; VP7:3), and Wa x ST3 (VP4:1A; VP7:4). One cold-adapted strain D is currently undergoing phase I studies in human volunteers. We investigated the role of specific rotavirus gene(s) of the cold-adapted (30 degrees Celsius) strain D involved in conferring (i) the cold-adaptation (ca) phenotype, i.e., capacity to replicate efficiently in vitro at 30 degrees Celsius, a temperature restrictive for wild-type virus, and (ii) the temperature sensitivity (ts) phenotype, i.e., restriction of replication in cell culture at 39 degrees Celsius in contrast to wild-type HRV which replicates efficiently at temperatures at 39 degrees Celsius. To identify the gene(s) associated with the ca and ts phenotypes, we generated reassortants by co-infection of AGMK or MA104 cells with the ca (30 degrees Celsius) and ts mutant of HRV strain D and wild-type HRV strain DS-1 (non-cold-adapted, non-temperature sensitive) at 33 degrees Celsius, a temperature permissive for both parental viruses. Fifty distinct reassortants were selected and plaque-purified at 33 degrees Celsius, the temperature used to generate the reassortants. The parental origin of each rotavirus gene segment was assigned for each reassortant by PAGE. The efficiency of plaque formation of parental and reassortant viruses in MA104 cells at 30, 37, and 39 degrees Celsius will be determined. In addition, a mutant of the 30 degrees Celsius-derived mutant that can plaque efficiently at 28 degrees Celsius was selected during successive serial passage at 28 degrees Celsius.